Studies have been devoted to determining how the activity of adenylate cyclase might be altered and regulated in cultured fibroblasts. Studies on the desensitization of hormonal responsiveness indicate that the rapid loss in sensitivity to catecholamines is not due to a loss of beta-adrenergic receptors. Rather, desensitization seems to involve the rapid uncoupling of the beta-receptor from adenylate cyclase. It was also observed that treatment of intact cells with quinacrine completely blocks, while treatment with tumor promoter (TPA) enhances, the isoproterenol-induced desensitization of the cyclase. These results suggest the possible involvement of phospholipase A2 in the desensitization process. Treatment with cholera toxin converts the cyclase system to a highly activated state which is sensitive to inhibition by GTP. Brief protease treatment of the choleragen activated membrane with papain negates the guanine nucleotide inhibitory response. This proteolytic effect is selective in that GTP inhibition is not eliminated by protease treatment of sparsely populated, rapidly growing cells. The results suggest that modulation of GTP stimulatory versus GTP inhibitory responses could serve an important regulatory role in controlling cyclic AMP production.